Gene therapy by secretory gland expression

ABSTRACT

Secretory gland cells, particularly pancreatic and salivary gland cells, are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subject. The expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein. The transformed secretory gland cells provide long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/410,660, filed Mar. 24, 1995.

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of gene therapy andmore particularly to the application of gene therapy to the cells of asecretory gland.

BACKGROUND OF THE INVENTION

[0003] Although gene therapy and specifically human gene therapy hasbeen widely discussed only over the last five years, the basic ideafirst became a reality in 1944 when Avery et al. carried out research onthe chemical nature of substances inducing transformation ofpneumococcal types. (Avery et al., J. Exp. Med. 79:137-158, 1944). Thework carried out by Avery et al., did not involve the actual insertionof genetic material into cells in order to carry out gene therapy. Theinsertion of new genetic material into cells in order to permanentlyaffect the genetic makeup of the cells is the methodology now generallyreferred to as gene therapy.

[0004] Current gene therapy is carried out in a variety of ways butinvolves two general protocols. In the first method, referred to as exvivo gene therapy, cells are extracted from an organism such as a humanand subsequently subjected to genetic manipulation by a variety ofdifferent means. After genetic material has been properly inserted intothe cells, the cells are implanted back into the body from which theywere removed. Thus the process involves cell removal, transformation ofthe cells in vitro, and subsequent reintroduction of the modified cellsinto the patient. Persistent, in vivo expression of the newly implantedgenetic material after transplantation of the transformed cells has beensuccessful (see Morgan et al., Science 237:1476 (1987); and Gerrard etal., Nat. Genet. 3:180 (1993)).

[0005] In the second approach to gene therapy, referred to as in vivogene therapy, somatic cells within a living organism are transformedwith new genetic material. For example, the genetic material to beintroduced into the organism is packaged within a retrovirus oradenovirus. The virus containing the desired genetic material is allowedto infect target cells within the organism. Upon infection of the cells,the virus injects genetic material into the cells which is thenintegrated into the cells' genome. As a result, the injected geneticmaterial is expressed and the patient is treated.

[0006] Several different methods for transforming cells can be used inaccordance with either the ex vivo or in vivo transfection procedures.For example, various mechanical methods can be used to deliver thegenetic material, including the use of fusogenic lipid vesicles(liposomes incorporating cationic lipids such as lipofection; seeFelgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987));direct injection of DNA (Wolff, et al., Science (1990) 247:1465-1468);and pneumatic delivery of DNA-coated gold particles with a devicereferred to as the gene gun (Yang et al., Proc. Natl. Acad. Sci. U.S.A.1990; 87:1568-9572).

[0007] Ex vivo and in vivo gene transfer methodologies have beenaccomplished using a variety of different procedures, such as the use ofretroviruses or direct injection. The procedures have been used on fivegeneral types of cells in order to carry out (1) liver cell genetherapy; (2) hematopoietic cell gene therapy; (3) cancer cell genetherapy; (4) respiratory cell gene therapy; and (5) muscle cell genetherapy. A review of the different techniques along with a citation ofnumerous publications in each area is contained within a recent articleon human gene therapy (see Morsy et al., JAMA 270:2338-2345 (1993)).

[0008] Depending on the desired result, the effect which the insertedgenetic material will have on the transformed cell can vary greatly andcan be selected according to the specific therapeutic situation. Forexample, genetic material inserted into the cells in order to obtaincirculation of the expressed genetic products would not be used inconnection with the treatment of cancer cells of a localized tumor.Stated differently, gene therapy may be carried out in order to locallyaffect a given type of cells such as affecting cancer cells within atumor or locally affecting liver cells. Other types of gene therapy arecarried out in order to cause the manipulated cells to express a proteinwhich is transported to the circulatory system and systemicallydelivered to the organism. Genetic manipulation of cells to express aprotein for systemic delivery to the organism has been problematic. Thepresent invention addresses this problem.

SUMMARY OF THE INVENTION

[0009] Secretory gland cells are genetically altered to operativelyincorporate a gene which expresses a therapeutically effective protein.More particularly, cells of a salivary gland or the pancreas aregenetically altered to operatively incorporate DNA which when expressedproduces a protein which has a desired therapeutic effect on thepatient. The expressed protein is secreted directly into the bloodstream and/or into the gastrointestinal system to obtain therapeuticlevels of the protein, thereby treating the patient in need of theprotein.

[0010] A primary object is to provide a method of gene therapy whereincells of a secretory gland, preferably the pancreas or a salivary gland,more preferably a parotid gland, of a mammal are genetically modified toexpress a biologically active and therapeutically useful protein whichprotein is secreted into the circulatory system and/or thegastrointestinal tract of the individual.

[0011] Another object is to produce genetically transformed secretorygland cells which cells have incorporated into their genome geneticmaterial which expresses a biologically active and therapeuticallyuseful protein and secretes that protein into the surrounding media.

[0012] An advantage of the present invention is that long termtherapeutic cures can be provided for diseases wherein individuals aresuffering from the disease due to a deficiency in a particular protein.A feature of the present invention is that cells of a secretory gland,preferably the pancreas or a salivary gland, more preferably a parotidgland, are specifically targeted.

[0013] Another advantage of the present invention is that the expressedprotein is secreted into the circulatory system and saliva or pancreaticjuices of the patient to provide a systemic effect.

[0014] These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the vectors, cell lines and methodology as morefully set forth below.

BRIEF DESCRIPTION OF THE DRAWING

[0015]FIG. 1 is a schematic view of a recombinant plasmid constructuseful in producing recombinant salivary gland cells of the invention;

[0016]FIG. 2 is a schematic view of a recombinant viral construct usefulin producing recombinant secretory gland cells of the invention;

[0017]FIGS. 3A and 3B are a schematic flow diagrams of production ofrecombinant secretory gland cells and their use in a therapeutic methodof the invention;

[0018]FIG. 4 is a map of the pFGH construct, which contains the humangrowth hormone genomic sequence;

[0019]FIG. 5 is a map of the pFGH.CMV construct, which contains thehuman growth hormone genomic sequence operably linked to the CMVpromoter;

[0020]FIG. 6 is a map of the pFGH.chymo construct, which contains thehuman growth hormone genomic sequence operably linked to thechymotrypsin B promoter;

[0021]FIG. 7 is a graph showing the levels of tissue expression of humangrowth hormone expression in the pancreas of rats after retrogradeinjection with either a control containing no DNA or a test samplecontaining a human growth hormone construct;

[0022]FIG. 8 is a graph showing the serum levels of human growth hormonein rats after retrograde pancreatic injection with either a controlcontaining no DNA or a test sample containing a human growth hormoneconstruct; and

[0023]FIG. 9 is a graph showing the correlation between pancreatictissue expression and serum levels of human growth hormone.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Before the present method of genetically transforming secretorygland cells and methods for providing gene therapy are described, it isto be understood that this invention is not limited to the particularmethodology, protocols, cell lines, secretory glands, vectors andreagents described as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

[0025] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a secretory gland cell” includes a plurality of such cells andreference to “the transformation vector” includes reference to one ormore transformation vectors and equivalents thereof known to thoseskilled in the art, and so forth.

[0026] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0027] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the cell lines,vectors, and methodologies which are described in the publications whichmight be used in connection with the presently described invention.

[0028] Definitions

[0029] By “secretory gland” is meant an aggregation of cells specializedto secrete or excrete materials not related to their ordinary metabolicneeds. Secretory glands include salivary glands, pancreas, mammaryglands, thyroid gland, thymus gland, pituitary gland, liver, and otherglands well known in the art.

[0030] By “salivary gland” is meant a gland of the oral cavity whichsecretes saliva, including the glandulae salivariae majores of the oralcavity (the parotid, sublingual, and submandibular glands) and theglandulae salivariae minores of the tongue, lips, cheeks, and palate(labial, buccal, molar, palatine, lingual, and anterior lingual glands).

[0031] By “pancreas” is meant a large, elongated, racemose glandsituated transversely behind the stomach, between the spleen and theduodenum. The pancreas is composed of an endocrine portion (the parsendocrina) and an exocrine portion (the pars exocrina). The parsendocrina, which contains the islets of Langerhans, produces andsecretes proteins, including insulin, directly into the blood stream.The pars exocrina contains secretory units and produces and secretes apancreatic juice, which contains enzymes essential to protein digestion,into the duodenum.

[0032] By “transformation” is meant a permanent genetic change inducedin a cell following incorporation of new DNA (i.e., DNA exogenous to thecell). Where the cell is a mammalian cell, the permanent genetic changeis generally achieved by introduction of the DNA into the genome of thecell.

[0033] By “transfection” is meant the transformation of a cell with DNAfrom a virus.

[0034] By “transformed cell” is meant a cell into which (or into anancestor of which) has been introduced, by means of recombinant DNAtechniques, a DNA molecule encoding a protein of interest.

[0035] By “DNA of interest” is meant any DNA sequence which encodes aprotein or other molecule which is desirable for administration,particularly intravenous administration, to a mammalian subject by genetherapy the sequence is generally operatively linked to other sequenceswhich are needed for its expression such as a promoter.

[0036] By “vector” is meant any compound, biological or chemical, whichfacilitates transformation of a target cell (e.g., a secretory glandcell) with a DNA of interest. Exemplary biological vectors includeviruses, particularly attenuated and/or replication-deficient viruses.Exemplary chemical vectors include lipid complexes and naked DNAconstructs.

[0037] By “promoter” is meant a minimal DNA sequence sufficient todirect transcription. “Promoter” is also meant to encompass thosepromoter elements sufficient for promoter-dependent gene expressioncontrollable for cell-type specific, tissue-specific or inducible byexternal signals or agents; such elements may be located in the 5′ or 3′regions of the native gene.

[0038] By “secretory gland specific promoter” is meant a promoter whichdirects expression of an operably linked DNA sequence when bound bytranscriptional activator proteins, or other regulators oftranscription, which are unique to a specific type of secretory glandcell. For example, by “salivary gland specific promoter” is meant asecretory gland specific promoter which directs expression in a salivarygland cell. A salivary amylase promoter is an example of a salivarygland specific promoter. By “pancreas specific promoter” is meant asecretory gland specific promoter which directs expression in apancreatic cell. Examples of pancreas specific promoters include apancreatic amylase promoter and an insulin promoter.

[0039] By “operably linked” is meant that a DNA sequence and aregulatory sequence(s) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

[0040] By “operatively inserted” is meant that the DNA of interestintroduced into the cell is positioned adjacent a DNA sequence whichdirects transcription and translation of the introduced DNA (i.e.,facilitates the production of, e.g., a polypeptide encoded by a DNA ofinterest).

[0041] By “mammalian subject” or “mammalian patient” is meant any mammalfor which gene therapy is desired, including human, bovine, equine,canine, and feline subjects.

[0042] The present invention features compositions and methods oftreatment using gene therapy, more specifically gene therapy byexpression of a DNA of interest in cells within a secretory gland of amammalian patient. Preferably, the transformed secretory gland cellsexpressing the protein encoded by the DNA of interest secrete atherapeutically effective amount of the protein into the blood stream orinto the gastrointestinal tract (e.g., into the saliva or pancreaticjuices) of the mammalian patient. Preferably, the secretory gland intowhich the DNA of interest is introduced and expressed is the pancreas,more preferably a salivary gland, even more preferably the parotidgland. Preferably, the DNA of interest encodes either insulin, a growthhormone, clotting factor VIII, intrinsic factor, or erythropoietin.Preferably, the DNA of interest is operably linked to a secretorygland-specific promoter. Where the secretory gland is the pancreas, thepromoter is preferably a pancreatic amylase promoter or an insulinpromoter. Where the secretory gland is a salivary gland, the promoter ispreferably a salivary amylase promoter.

[0043] The invention also features recombinant secretory gland cells,preferably recombinant pancreatic or recombinant salivary gland cells,more preferably recombinant parotid gland cells, containing a DNA ofinterest operatively inserted in the genome of the cell and operativelylinked to a promoter for expression of the DNA of interest. Preferably,the promoter operatively linked to the DNA of interest is a secretorygland specific promoter. Where the secretory gland is the pancreas, thepromoter is preferably a pancreatic amylase promoter or insulinpromoter. Where the secretory gland is a salivary gland, the promoter ispreferably a salivary amylase promoter.

[0044] The invention will now be described in further detail.

[0045] Vectors and Constructs

[0046] Any nucleic acid vector having a eukaryotic promoter operablylinked to a DNA of interest can be used in the invention to transform asecretory gland cell. The vectors containing the DNA sequence (or thecorresponding RNA sequence) which may be used in accordance with theinvention may be any eukaryotic expression vector containing the DNA orthe RNA sequence of interest. For example, a plasmid or viral vector(e.g. adenovirus) can be cleaved to provide linear DNA having ligatabletermini. These termini are bound to exogenous DNA having complementary,like ligatable termini to provide a biologically functional recombinantDNA molecule having an intact replicon and a desired phenotypicproperty.

[0047] A variety of techniques are available for DNA recombination inwhich adjoining ends of separate DNA fragments are tailored tofacilitate ligation. The exogenous (i.e., donor) DNA used in theinvention is obtained from suitable cells. The vector is constructedusing known techniques to obtain a transformed cell capable of in vivoexpression of the therapeutic protein. The transformed cell is obtainedby contacting a target cell with a RNA- or DNA-containing formulationpermitting transfer and uptake of the RNA or DNA into the target cell.Such formulations include, for example, viruses, plasmids, liposomalformulations, or plasmids complexed with polycationic substances such aspoly-L-lysine or DEAC-dextran, and targeting ligands.

[0048] Techniques for obtaining expression of exogenous DNA or RNAsequences in a host are known in the art (see, for example, Kormal etal., Proc. Natl. Acad. Sci. USA, 84:2150-2154, 1987; Sambrook et al.Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; each of which arehereby incorporated by reference with respect to methods andcompositions for eukaryotic expression of a DNA of interest).

[0049] Various vectors (e.g., viral vectors, bacterial vectors, orvectors capable of replication in eukaryotic and prokaryotic hosts) canbe used in accordance with the present invention. Preferably the vectoris capable of replication in both eukaryotic and prokaryotic hosts.Numerous vectors which can replicate in eukaryotic and prokaryotic hostsare known in the art and are commercially available. In general, suchvectors used in accordance with the invention are composed of abacterial origin of replication and a eukaryotic promoter operablylinked to a DNA of interest.

[0050] In general, viral vectors used in accordance with the inventionare composed of a viral particle derived from a naturally-occurringvirus which has been genetically altered to render the virusreplication-defective and to express a recombinant gene of interest inaccordance with the invention. FIG. 3 shows a schematic view of anexemplary recombinant vector construct useful in the method of theinvention. Once the virus delivers its genetic material to a cell, itdoes not generate additional infectious virus but does introduceexogenous recombinant genes into the cell, preferably into the genome ofthe cell.

[0051] Numerous viral vectors are well known in the art, including, forexample, retrovirus, adenovirus, adeno-associated virus, herpes simplexvirus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors.Retroviral vectors are less preferred since retroviruses requirereplicating cells and secretory glands are composed of mostly slowlyreplicating and/or terminally differentiated cells. Adenovirus is apreferred viral vector since this virus efficiently infects slowlyreplicating and/or terminally differentiated cells. Where the secretorygland is a salivary gland, the viral vector is preferably derived froman attenuated and/or replication-deficient mumps virus or otherattenuated and/or replication-deficient virus which is substantiallyspecific for salivary gland cells.

[0052] Where a replication-deficient virus is used as the viral vector,the production of infective virus particles containing either DNA or RNAcorresponding to the DNA of interest can be produced by introducing theviral construct into a recombinant cell line which provides the missingcomponents essential for viral replication in trans. Preferably,transformation of the recombinant cell line with the recombinant viralvector will not result in production of replication-competent viruses,e.g., by homologous recombination of the viral sequences of therecombinant cell line into the introduced viral vector.

[0053] Methods for production of replication-deficient viral particlescontaining a DNA of interest are well known in the art and are describedin, for example, Rosenfeld et al., Science 252:431-434, 1991 andRosenfeld et al., Cell 68:143-155, 1992 (adenovirus); U.S. Pat. No.5,139,941 (adeno-associated virus); U.S. Pat. No. 4,861,719(retrovirus); and U.S. Pat. No. 5,356,806 (vaccinia virus). Methods andmaterials for manipulation of the mumps virus genome, characterizationof mumps virus genes responsible for viral fusion and viral replication,and the structure and entire sequence of the mumps viral genome aredescribed in Tanabayashi et al., J. Virol. 67:2928-2931, 1993; Takeuchiet al., Archiv. Virol., 128:177-183, 1993; Tanabayashi et al., Virol.187:801-804, 1992; Kawano et al., Virol., 179:857-861, 1990; Elango etal., J. Gen. Virol. 69:2893-28900, 1988. Given the knowledge in the artregarding the mumps viral genome and the genes important for mumps virusfusion and replication, mumps viral vectors can be readily constructed,and replication defective mumps virus strains developed, for use insalivary gland specific gene transfer, gene expression and gene therapy.

[0054] The DNA of interest may be administered using a non-viral vector,for example, as a DNA- or RNA-liposome complex formulation. Suchcomplexes comprise a mixture of lipids which bind to genetic material(DNA or RNA), providing a hydrophobic coat which allows the geneticmaterial to be delivered into cells. Liposomes which can be used inaccordance with the invention include DOPE (dioleyl phosphatidyl ethanolamine), CUDMEDA (N-(5-cholestrum-3-β-ol3-urethanyl)-N′,N′-dimethylethylene diamine). When the DNA of interestis introduced using a liposome, it is preferable to first determine invitro the optimal values for the DNA: lipid ratios and the absoluteconcentrations of DNA and lipid as a function of cell death andtransformation efficiency for the particular type of cell to betransformed. These values can then be used in or extrapolated for use inin vivo transformation. The in vitro determinations of these values canbe readily carried out using techniques which are well known in the art.

[0055] Other non-viral vectors may also be used in accordance with thepresent invention. These include chemical formulations of DNA or RNAcoupled to a carrier molecule (e.g., an antibody or a receptor ligand)which facilitates delivery to host cells for the purpose of altering thebiological properties of the host cells. By the term “chemicalformulations” is meant modifications of nucleic acids to allow couplingof the nucleic acid compounds to a carrier molecule such as a protein orlipid, or derivative thereof. Exemplary protein carrier moleculesinclude antibodies specific to the cells of a targeted secretory glandor receptor ligands, i.e., molecules capable of interacting withreceptors associated with a cell of a targeted secretory gland.

[0056] Preferably, the DNA construct contains a promoter to facilitateexpression of the DNA of interest within a secretory gland cell, morepreferably a pancreatic cell or salivary gland cell, even morepreferably a parotid gland cell. Preferably the promoter is a strong,eukaryotic promoter. Exemplary eukaryotic promoters include promotersfrom cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Roussarcoma virus (RSV), and adenovirus. More specifically, exemplarypromoters include the promoter from the immediate early gene of humanCMV (Boshart et al., Cell 41:521-530, 1985) and the promoter from thelong terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci.USA 79:6777-6781, 1982). Of these two promoters, the CMV promoter ispreferred as it provides for higher levels of expression than the RSVpromoter.

[0057] Where the secretory gland is the pancreas, the promoter used inthe vector is preferably a pancreas specific promoter, i.e. the promoterfacilitates specific expression of the DNA to which it is operablylinked when the construct is in the presence of a pancreas cell specifictranscriptional activator protein. The promoters may be derived from thegenome of any mammal, and is preferably derived from a murine or a humansource, more preferably from a human source. Examples of preferredpancreas specific promoters include the insulin promoter and pancreasα-amylase promoters.

[0058] Where the secretory gland is a salivary gland, the promoter usedin the vector is preferably a salivary gland specific promoter, i.e. thepromoter facilitates specific expression of the DNA to which it isoperably linked when the construct is in the presence of a salivarygland cell specific transcriptional activator protein(s). Examples ofpreferred salivary gland specific promoters include salivary α-amylasepromoters and mumps viral gene promoters which are specificallyexpressed in salivary gland cells.

[0059] Multiple pancreatic α-amylase genes and multiple salivaryα-amylase genes, have been identified and characterized in both mice andhumans (see, for example, Jones et al., Nucleic Acids Res.,17:6613-6623; Pittet et al., J. Mol. Biol., 182:359-365, 1985;Hagenbuchle et al., J. Mol. Biol., 185:285-293, 1985; Schibler et al.,Oxf. Surv. Eukaryot. Genes, 3:210-234, 1986; and Sierra et al., Mol.Cell. Biol., 6:4067-4076, 1986 for murine pancreatic and salivaryα-amylase genes and promoters; Samuelson et al., Nucleic Acids Res.,16:8261-8276, 1988; Groot et al., Genomics, 5:29-42, 1989; and Tomita etal., Gene, 76:11-18, 1989 for human pancreatic and salivary α-amylasegenes and their promoters). The promoters of these α-amylase genesdirect either pancreas or salivary gland specific expression of theircorresponding α-amylase encoding DNAs. These promoters may thus be usedin the constructs of the invention to achieve pancreas-specific orsalivary gland-specific expression of a DNA of interest.

[0060] For example, the human genome contains three, nearly identicalsalivary α-amylase genes, termed AMYlA, AMYLB, and AMYlC, as well as atleast two pancreatic α-amylase genes. The promoters of three salivaryα-amylase genes appear identical. The region from −1003 to −327 basepairs of the human salivary α-amylase AMYLC promoter sequence issufficient for parotid gland specific expression, while the region from−1003 to −826 is necessary for salivary gland specific expression (Tinget al., Genes Dev. 6:1457-65, 1992). This human salivary promotersequence can be operably linked to a DNA of interest in a construct forsalivary-gland specific expression according to the present invention.

[0061] Recombinant promoters derived from any of the above-describedpromoters may also be employed in constructs to achieve secretory glandspecific expression of a specific gene of interest. For example, arecombinant promoter capable of directing expression at increased levelsrelative to a wild-type salivary α-amylase promoter may be produced bymultimerizing the −1003 to −826 region of the salivary α-amylasepromoter in the construct, or by operably linking a viral enhancersequence (e.g., an enhancer sequence from CMV) to a full length salivaryα-amylase promoter sequence.

[0062] The constructs of the invention may also include sequences inaddition to promoters which enhance secretory gland specific expression.For example, where pancreas specific expression of the DNA of interestis desired, the construct may include a PTF-1 recognition sequence(Cockell et al., Mol. Cell. Biol., 9:2464-2476, 1989). Sequences whichenhance salivary gland specific expression are also well known in theart (see, for example, Robins et al., Genetica 86:191-201, 1992).

[0063] Other components such as a marker (e.g., an antibiotic resistancegene (such as an ampicillin resistance gene) or β-galactosidase) to aidin selection of cells containing and/or expressing the construct, anorigin of replication for stable replication of the construct in abacterial cell (preferably, a high copy number origin of replication), anuclear localization signal, or other elements which facilitateproduction of the DNA construct, the protein encoded thereby, or both.

[0064] For eukaryotic expression (e.g., in a salivary gland cell), theconstruct should contain at a minimum a eukaryotic promoter operablylinked to a DNA of interest, which is in turn operably linked to apolyadenylation sequence. The polyadenylation signal sequence may beselected from any of a variety of polyadenylation signal sequences knownin the art. Preferably, the polyadenylation signal sequence is the SV40early polyadenylation signal sequence. The construct may also includeone or more introns, which can increase levels of expression of the DNAof interest, particularly where the DNA of interest is a CDNA (e.g.,contains no introns of the naturally-occurring sequence). Any of avariety of introns known in the art may be used. Preferably, the intronis the human β-globin intron and inserted in the construct at a position5′ to the DNA of interest.

[0065] The DNA of interest can be any DNA encoding any protein for whichintravenous therapy and/or therapy for the gastrointestinal tract isdesirable. For example, intravenous protein therapy is appropriate intreating a mammalian subject having an inherited or acquired diseaseassociated with a specific protein deficiency (e.g., diabetes,hemophilia, anemia, severe combined immunodeficiency). Such proteindeficient states are amenable to treatment by replacement therapy, i.e.,expression of a protein to restore the normal blood stream levels of theprotein to at least normal levels. Secretion of a therapeutic protein tothe gastrointestinal tract (e.g. by secretion of the protein into thesaliva, pancreatic juices, or other mucosal secretion) is appropriatewhere, for example, the subject suffers from a protein deficiencyassociated with absorption of nutrients (e.g. deficiency in intrinsicfactor) or digestion (e.g., deficiencies in various pancreatic enzymes).

[0066] Alternatively, the mammalian subject may have a condition whichis amenable to treatment by expression or over-expression of a proteinwhich is either normally present in a healthy mammalian subject or isforeign to the mammalian subject. For example, intravenous proteintherapy can be used in treatment of a mammalian subject having a viral(e.g., human immunodeficiency virus (HIV), Epstein-Barr virus (EBV),herpes simplex virus (HSV), bacterial, fungal, and/or parasiticinfection, particularly where the infection is chronic, i.e., persistingover a relatively long period of time. The secretory gland gene therapyof the invention may also be used to enhance expression of a proteinpresent in a normal mammal, or to express a protein not normally presentin a normal mammal, in order to achieve a desired effect (e.g., toenhance a normal metabolic process). For example, a secretory gland of adairy cow may be transformed with DNA encoding bovine growth hormone(BGH) in order to enhance levels of BGH in the blood stream and enhancemilk-production.

[0067] The DNA of interest is preferably obtained from a source of thesame species as the mammalian subject to be treated (e.g. human tohuman), but this is not an absolute requirement. DNA obtained from aspecies different from the mammalian subject can also be used,particularly where the amino acid sequences of the proteins are highlyconserved and the xenogeneic protein is not highly immunogenic so as toelicit a significant, undesirable antibody response against the proteinin the mammalian host.

[0068] Exemplary, preferred DNAs of interest include DNA encodinginsulin, growth hormone, clotting factor VIII, intrinsic factor, anderythropoietin. Of particular interest is intravenous protein therapy ofa mammalian subject (e.g., a bovine, canine, feline, equine, or humansubject, preferably a bovine or human subject, more preferably a humansubject) by expression of DNA encoding a protein (e.g., insulin, growthhormone, clotting factor VIII, or erythropoietin) in a transformedmammalian salivary gland cell, preferably a mammalian parotid glandcell. Preferably, the subject is a human subject and the DNA expressedencodes a human protein (e.g., human insulin, human growth hormone,human clotting factor VIII, or human erythropoietin). Other exemplaryDNAs of interest include tissue plasminogen activator (tPA), urokinase,streptokinase, acidic fibroblast growth factor, basic fibroblast growthfactor, tumor necrosis factor alpha, tumor necrosis factor β,transforming growth factor β, platelet-derived growth factor,endothelian, and soluble CD4. Table 1 provides a list of exemplaryproteins and protein classes which can be delivered by the secretorygland gene therapy of the invention. TABLE 1 Exemplary Proteins andProtein Classes for Secretory Gland Gene Therapy SPECIFIC EXEMPLARYPROTEINS insulin interferon-α2B human growth hormone (hGH) transforminggrowth factor (TGF) erythropoietin (EPO) ciliary neurite transformingfactor (CNTF) clotting factor VIII insulin-like growth factor-1 (IGF-1)bovine growth hormone (BGH) granulocyte macrophage colony stim- ulatingfactor (GM-CSF) platelet derived growth factor interferon-α2A (PDGF)clotting factor VIII brain-derived neurite factor (BDNF) thrombopoietin(TPO) insulintropin IL-1 tissue plasminogen activator (tPA) IL-2urokinase IL-1 RA streptokinase superoxide dismutase (SOD) adenosinedeamidase catalase calcitonin fibroblast growth factor (FGF) arginase(acidic or basic) neurite growth factor (NGF) phenylalanine ammonialyase granulocyte colony stimulating γ-interferon factor (G-CSF)L-asparaginase pepsin uricase trypsin chymotrypsin elastasecarboxypeptidase lactase sucrase intrinsic factor calcitonin parathyroidhormone(PTH)- like hormone Ob gene product cholecystokinin (CCK)glucagon insulinotrophic hormone EXEMPLARY CLASSES OF PROTEINS proteasespituitary hormones protease inhibitors growth factors cytokinessomatomedians chemokines immunoglobulins gonadotrophins interleukinschemotactins interferons lipid-binding proteins

[0069] Various disease conditions are amenable to the treatment usingthe secretory gland gene therapy of the invention. One skilled in theart can recognize the appropriate protein which should be produced bythe invention for treating specific disease conditions. Exemplarydiseases which are amenable to treatment using the subject invention,and exemplary, appropriate proteins which can be used in treating thesediseases, are shown in Table 2. TABLE 2 Exemplary Disease ConditionsAmenable to Secretory Gland Gene Therapy Enzyme Deficiency EndotoxicShock/Sepsis Adenosine deaminase¹ Lipid-binding proein (LBP) Purinenucleotide phosphorylase Galactosidase β-glucuronidase Antioxidants forCancer Therapy Anemia Superoxide dismutase Erythropoietin CatalaseCancer Growth Factors (for use in wound α-Interferon healing, inductionof red blood γ-Interferon cell formation, etc.) α-IL1 Epidermal growthfactor Phenylalanine ammonia lyase G-CSF Arginase γ-InterferonL-asparaginase Transforming growth factor Uricase ErythropoietinGranulocyte colony stimulating Thrombopoietin factor (G-CSF)Insulin-like growth factor-1 Monoclonal antibodies Insulin Tissuenecrosis factor Human growth hormone Cardiovascular Disease DiabetesTissue plasminogen activator Insulin Urokinase (native or chimeric)Glucagon α₁-antitypsin Insulinotrophic hormone Antithrombin-III Otherproteases or protease inhibitors Clotting disorders Apolipoproteins(particularly B-48) Clotting factor VIII Circulating Scavenger ReceptorAPO A1₂ Obesity and Feeding Autoimmune diseases Ob gene productIntrinsic factor Cholecystokinin (CCK) (for pernicious anemia, a vitaminB₁₂ absorption deficiency) Bone diseases Gastrointestinal and PancreaticCalcitonin Deficiencies PTH-like hormone Pepsin (for esophageal reflux)Trypsin Chymotrypsin Elastase Carboxypeptidase Lactase (for lactosedeficiency) Sucrase Intrinsic Factor

[0070] Numerous proteins which are desirable for intravenous proteintherapy are well known in the art and the DNA encoding these proteinshas been isolated. For example, the sequence of the DNAs encodinginsulin, human growth hormone, intrinsic factor, clotting factor VIII,and erythropoietin are available from Genbank and/or have been describedin the scientific literature (e.g., human clotting factor VIII gene:Gitschier et al., Nature 312:326-330, 1984; Wood et al., Nature312:330-337, 1984; human intrinsic factor: Hewitt et al., Genomics10:432-440, 1991). Proteins commonly used in treatments can be used inthe gene therapy procedures of the present invention. Such proteins aredisclosed in, for example, the Physicians' Desk Reference (1994Physicians' Desk Reference, 48th Ed., Medical Economics Data ProductionCo., Montvale, N.J.; incorporated by reference) and can be dosed usingmethods described in Harrison's Principles of Internal Medicine and/orthe AMA “Drug Evaluations Annual” 1993, all incorporated by reference.

[0071] Where the DNA encoding a protein of interest has not beenisolated, this can be accomplished by various, standard protocols wellknown to those of skill in the art (see, for example, Sambrook et al.,ibid; Suggs et al., Proc. Natl. Acad. Sci. USA 78:6613-6617, 1981; U.S.Pat. No. 4,394,443; each of which are incorporated herein by referencewith respect to identification and isolation of DNA encoding a proteinof interest). For example, genomic or cDNA clones encoding a specificprotein can be isolated from genomic or CDNA libraries usinghybridization probes designed on the basis of the nucleotide or aminoacid sequences for the desired gene. The probes can be constructed bychemical synthesis or by polymerase chain reaction (PCR) using primersbased upon sequence data to amplify DNA fragments from pools orlibraries (U.S. Pat. Nos. 4,683,195 and 4,683,202). Nucleotidesubstitutions, deletions, additions, and the like can also beincorporated into the polynucleotides, so long as the ability of thepolynucleotide to hybridize is not substantially disrupted. (Sambrook etal. ibid). The clones may be expressed or the DNA of interest can beexcised or synthesized for use in other constructs. If desired, the DNAof interest can be sequenced using methods well known in the art.

[0072] In a preferred embodiment, the construct used in the presentinvention is designed so as to enhance protein secretion from thetransformed secretory gland cell into the blood stream. Secretory glandcells are normally polarized, with the apical surface oriented towardthe ductal system and the basolateral surface oriented toward the bloodsupply. Most proteins produced by the pancreas and salivary glands arereleased into the duct system and eventually into the gastrointestinaltract. However, some secretory gland proteins, such as kallikreins, aresecreted primarily into the blood stream. Regardless of whether aspecific secretory gland protein is primarily released into the ductsystem or into the blood stream, there is a modest rate of transport ofthese same proteins into the secondary system. Secretory gland proteinsare not normally partitioned solely into the blood stream or solely intothe gastrointestinal tract. For example, amylase, which is primarilysecreted into the duct systems, is also released at a lower level intothe blood stream.

[0073] The specific features responsible for mediatingintravenous-directed or duct system-directed secretion have not beendescribed. However, when salivary gland cells are transformed with DNAencoding insulin according to the present invention, relatively littleinsulin is released into the saliva as compared to the blood. Thisobservation suggests that the polypeptide itself contains theinformation for targeting of secretion.

[0074] Preferably, the DNA of interest contains a secretion signal whicheither directs secretion of the protein primarily into the duct systemor directs secretion of the protein primarily into the blood stream.Intravenous-directed secretion signals and duct system-directedsecretion signals can be identified by, for example, site-directedmutagenesis of DNA encoding a blood stream-targeted protein (e.g.,insulin) or a duct system-targeted protein (e.g., amylase). The mutantscan be screened by expression of the mutated DNA in secretory glandcells and subsequently determining the ratio of, for example, salivaryto intravenous expression. Alternatively, intravenous-directed secretionsignals and duct system-directed secretion signals can also beidentified by constructing recombinant, chimeric proteins composed of,for example, a putative intravenous secretion signal inserted into asaliva-directed protein. Intravenous secretion signals would then beidentified by their ability to re-direct expression of thesaliva-directed protein into the blood stream. Putative intravenoussecretion signals and duct system secretion signals can also beidentified by comparison of DNA and amino acid sequences of proteinswhich are preferentially secreted into either the blood stream or theduct system, respectively. Areas of homology or common motifs among theproteins could then be tested as described above.

[0075] The DNA of interest may be inserted into a construct so that thetherapeutic protein is expressed as a fusion protein (e.g., a fusionprotein having β-galactosidase or a portion thereof at the N-terminusand the therapeutic protein at the C-terminal portion). Production of afusion protein can facilitate identification of transformed cellsexpressing the protein (e.g., by enzyme-linked immunosorbent assay(ELISA) using an antibody which binds to the fusion protein).

[0076] It may also be desirable to produce altered forms of thetherapeutic proteins that are, for example, protease resistant or haveenhanced activity relative to the wild-type protein. For example, wherean enzyme is to be secreted into saliva or pancreatic juices, it may beadvantageous to modify the protein so that it is resistant to digestiveproteases. Further, where the therapeutic protein is a hormone, it maybe desirable to alter the protein's ability to form dimers or multimericcomplexes. For example, insulin modified so as to prevent itsdimerization has a more rapid onset of action relative to wild-type,dimerized insulin.

[0077] The construct containing the DNA of interest can also be designedso as to provide for site-specific integration into the genome of thetarget secretory gland cell. For example, a construct can be producedsuch that the DNA of interest and the promoter to which it is operablylinked are flanked by the position-specific integration markers ofSaccharomyces cerevisiae Ty3. The construct for site-specificintegration additionally contains DNA encoding a position-specificendonuclease which recognizes the integration markers. Such constructstake advantage of the homology between the Ty3 retrotransposon andvarious animal retroviruses. The Ty3 retrotransposon facilitatesinsertion of the DNA of interest into the 5′ flanking region of manydifferent tRNA genes, thus providing for more efficient integration ofthe DNA of interest without adverse effect upon the recombinant cellproduced. Methods and compositions for preparation of such site-specificconstructs are described in U.S. Pat. No. 5,292,662, incorporated hereinby reference with respect to the construction and use of suchsite-specific insertion vectors.

[0078] Transformation

[0079] Introduction of the DNA of interest into the genome of thesecretory gland cell can be accomplished by various methods well knownin the art. For example, transformation of secretory gland cells can beaccomplished by administering the DNA of interest directly to themammalian subject (in vivo gene therapy), or to a in vitro culture of abiopsy of secretory glands cells which are subsequently transplantedinto the mammalian subject after transformation (ex vivo gene therapy).

[0080] The DNA of interest can be delivered to the subject or the invitro cell culture as, for example, purified DNA, in a viral vector(e.g., adenovirus, mumps virus, retrovirus), a DNA- or RNA-liposomecomplex, or by utilizing cell-mediated gene transfer. Further, thevector, when present in non-viral form, may be administered as a DNA orRNA sequence-containing chemical formulation coupled to a carriermolecule which facilitates delivery to the host cell. Such carriermolecules can, for example, include an antibody specific to an antigenexpressed on the surface of the targeted secretory gland cells, or someother molecule capable of interaction with a receptor associated withsecretory gland cells.

[0081] The DNA or RNA sequence encoding the molecule used in accordancewith the invention may be either locally or systemically administered tothe mammalian subject, which may be human or a non-human mammal (e.g.,bovine, equine, canine, feline). Where the targeted secretory gland is asalivary gland local administration is preferably by injection into ornear a salivary gland or by retrograde perfusion of a salivary glandduct system. More preferably the salivary gland is a parotid gland.Where the targeted secretory gland is the pancreas, local administrationis preferably by cannulation of the pancreatic duct by duodenalintubation, using endoscopic retrograde chalangio-pancreatography(ECRP).

[0082] Systemic administration can be carried out by intramuscularinjection of a viral vector containing the DNA of interest. Where thetargeted secretory gland is a salivary gland, systemic administration ispreferably by oral administration of a viral vector containing a DNA ofinterest, preferably a adenovirus vector, more preferably a mumps virusvector or other virus vector which substantially specifically infectscells of the salivary gland. Where the targeted secretory gland is thepancreas, systemic administration is preferably achieved byadministration of the DNA of interest in a viral vector orDNA-containing formulation (e.g. liposome) which binds thecholecystokinin (CCK) receptor.

[0083] As indicated above, the secretory gland cells of a patient may betransformed ex vivo by collecting a biopsy of the secretory glandtissue, culturing secretory gland cells from the biopsy in vitro, andtransfecting the cultured secretory gland cells with a DNA of interestin vitro. The resulting transformed secretory gland cells are thenimplanted into the mammalian subject, preferably into the correspondingsecretory gland of the mammalian subject from which the biopsy wastaken. Preferably, the secretory gland cells are transformed in vivo byeither mechanical means (e.g., direct injection of the DNA of interestinto or in the region of the secretory gland or lipofection) or bybiological means (e.g., infection of a salivary gland with anon-pathogenic virus, preferably a non-replicative virus, containing theDNA of interest). More preferably, the salivary gland cells aretransformed in vivo by infection with a non-replicative virus containingthe DNA of interest.

[0084] The form of the preparation for transformation of the secretorygland cells will depend upon several factors such as whethertransformation is performed ex vivo or in vivo, the secretory glandtargeted for gene transfer, the route of administration, and whether abiological or non-biological vector is employed. For example, where thepreparation for transformation is administered via the oral route, thepreparation may be formulated to provide mucosal resistance (e.g.,resistance to proteolytic digestion, denaturation in the mucosalenvironment, etc.). In addition to the DNA of interest, such oralpreparations can include detergents, gelatins, capsules, or otherdelivery vehicles to protect against degradation.

[0085] Generally, transformation is accomplished by either infection ofthe secretory gland cells with a virus, preferably areplication-deficient virus, containing the DNA of interest, or by anon-viral transformation method, such as direct injection of the DNAinto or near the target salivary gland cell, lipofection, “gene gun”, orother methods well known in the art. The preferred methodology isdependent upon whether the gene transfer is performed ex vivo or invivo.

[0086] Ex vivo secretory gland gene therapy is accomplished by obtaininga biopsy of tissue from a secretory gland and establishing a primaryculture of these secretory gland cells. Methods for obtaining salivarygland tissue biopsy and growing cells from this tissue in vitro are wellknown in the art. Methods for separation of cells from tissue (see, forexample, Amsterdam et al., J. Cell Biol. 63:1057-1073, 1974), andmethods for culturing cells in vitro are well known in the art.

[0087] The secretory gland cells in the in vitro culture are thentransformed using various methods known in the art. For example,transformation can be performed by calcium or strontium phosphatetreatment, microinjection, electroporation, lipofection, or viralinfection. For example, the cells may be injected with a moloney-LTRdriven construct or lipofected with an adenovirus-, vaccinia virus-,HIV-, or CMV-promoter construct. The transfected DNA plasmid can containa selectable marker gene or be co-transfected with a plasmid containinga selectable marker.

[0088] Where one or more selectable markers are transferred into thecells along with the DNA of interest, the cell populations containingthe DNA of interest can be identified and enriched by selecting for themarker(s). Typically markers provide for resistance to antibiotics suchas tetracycline, hygromycin, neomycin, and the like. Other markers caninclude thymidine kinase and the like.

[0089] The ability of the transformed secretory gland cells to expressthe DNA of interest can be assessed by various methods known in the art.For example, the ability of the cells to secrete the protein into thecell culture media can be examined by performing an ELISA on a sample ofcell culture supernatant using an antibody which specifically binds theprotein encoded by the DNA of interest. Alternatively, expression of theDNA of interest can be examined by Northern blot to detect mRNA whichhybridizes with a DNA probe derived a selected sequence of the DNA ofinterest. Those cells which express the protein encoded by the DNA ofinterest can be further isolated and expanded in in vitro culture usingmethods well known in the art.

[0090] After expansion of the transformed secretory gland cells invitro, the cells are implanted into the mammalian subject, preferablyinto the secretory gland from which the cells were originally derived,by methods well known in the art. Preferably the cells are implanted inan area of dense vascularization, and in a manner that minimizesevidence of surgery in the subject. The engraftment of the implant oftransformed secretory gland cells is monitored by examining themammalian subject for classic signs of graft rejection, i.e.,inflammation and/or exfoliation at the site of implantation, and fever.

[0091] In vivo transformation methods normally employ either abiological means of introducing the DNA into the target cells (e.g., avirus containing the DNA of interest) or a mechanical means to introducethe DNA into the target cells (e.g., direct injection of DNA into thecells, liposome fusion, pneumatic injection using a “gene gun”).Generally the biological means used for in vivo transformation of targetcells is a virus, particularly a virus which is capable of infecting thetarget cell, and integrating at least the DNA of interest into thetarget cell's genome, but is not capable of replicating. Such virusesare referred to as replication-deficient viruses orreplication-deficient viral vectors. Alternatively, the virus containingthe DNA of interest is attenuated, i.e. does not cause significantpathology or morbidity in the infected host (i.e., the virus isnonpathogenic or causes only minor disease symptoms).

[0092] Numerous viral vectors useful in in vivo transformation and genetherapy are known in the art, or can be readily constructed given theskill and knowledge in the art. Exemplary viruses includenon-replicative mutants/variants of adenovirus, mumps virus, retrovirus,adeno-associated virus, herpes simplex virus (HSV), cytomegalovirus(CMV), vaccinia virus, and poliovirus. Preferably, thereplication-deficient virus is capable of infecting slowly replicatingand/or terminally differentiated cells, since secretory glands areprimarily composed of these cell types. Thus, adenovirus is a preferredviral vector since this virus efficiently infects slowly replicatingand/or terminally differentiated cells. More preferably, the viralvector is specific or substantially specific for cells of the targetedsecretory salivary gland. For example, the mumps virus is particularlypreferred where the targeted secretory gland is a salivary gland.

[0093] In vivo gene transfer using a biological means can beaccomplished by administering the virus containing the DNA to themammalian subject either by an intraductal route, an oral route, or byinjection depending upon the secretory gland targeted for gene transfer.The amount of DNA and/or the number of infectious viral particleseffective to infect the targeted secretory gland, transform a sufficientnumber of secretory gland cells, and provide for expression oftherapeutic levels of the protein can be readily determined based uponsuch factors as the efficiency of the transformation in vitro, thelevels of protein expression achieved in vitro, and the susceptibilityof the targeted secretory gland cells to transformation. For example,where the targeted secretory gland is a salivary gland and where a viruscontaining the DNA of interest is administered orally, the virus will beadministered at a concentration effective to infect salivary gland cellsof the mammalian subject and provide for therapeutic levels of theprotein in either the blood or the saliva.

[0094] Various mechanical means can be used to introduce a DNA ofinterest directly into a secretory gland for expression in a secretorygland cell of a mammalian subject. For example, the DNA of interest maybe introduced into a salivary gland by percutaneous injection or byretrograde injection via the ducts leading from the oral mucosa to thesalivary gland. Preferably, the DNA is injected percutaneously into theparotid gland of the mammalian subject. Where the secretory gland is thepancreas, direct administration of the DNA of interest into the pancreascan be accomplished by cannulation of the pancreatic duct by, forexample duodenal intubation. Alternatively, administration of the viruscontaining the DNA of interest may be accomplished by intramuscularinjection.

[0095] The DNA of interest may be naked (i.e., not encapsulated),provided as a formulation of DNA and cationic compounds (e.g., dextransulfate), or may be contained within liposomes. Alternatively, the DNAof interest can be pneumatically delivered using a “gene gun” andassociated techniques which are well known in the art (Fynan et al.Proc. Natl. Acad. Sci. USA 90:11478-11482, 1993). Where the targetedsecretory gland is a salivary gland, the DNA of interest is preferablyintroduced by direct percutaneous injection of naked DNA into thesalivary gland, preferably into the parotid gland.

[0096] The amount of DNA administered will vary greatly according to anumber of factors including the susceptibility of the target cells totransformation, the size and weight of the subject, the levels ofprotein expression desired, and the condition to be treated. Forexample, the amount of DNA injected into a secretory gland of a human isgenerally from about 1 μg to 200 mg, preferably from about 100 μg to 100mg, more preferably from about 500 μg to 50 mg, most preferably about 10mg. The amount of DNA injected into the pancreas of a human is, forexample, generally from about 1 μg to 750 mg, preferably from about 500μg to 500 mg, more preferably from about 10 mg to 200 mg, mostpreferably about 100 mg. Generally, the amounts of DNA for human genetherapy can be extrapolated from the amounts of DNA effective for genetherapy in an animal model. For example, the amount of DNA for genetherapy in a human is roughly 100 times the amount of DNA effective ingene therapy in a rat. The amount of DNA necessary to accomplishsecretory gland cell transformation will decrease with an increase inthe efficiency of the transformation method used.

[0097] Intravenous and Gastrointestinal Protein TherapY byTransformation of Salivary Gland Cells

[0098] Secretory glands transformed according to the inventionfacilitate high level expression of a DNA of interest, particularlywhere the DNA of interest is operably linked to a strong eukaryoticpromoter (e.g., CMV, MMTV, or pancreatic or salivary amylase promoters).The expressed protein is then secreted at high levels into the bloodstream or into the gastrointestinal tract via saliva or pancreaticjuices. The protein so expressed and secreted is thus useful in treatinga mammalian subject having a variety of conditions. For example,secretion of an appropriate protein into the saliva is useful inpreventing or controlling various upper gastrointestinal tract diseases,e.g., in treating chronic infections of the oral cavity, (e.g.,bacterial or fungal infections); in treating degenerative disorders ofthe salivary glands, in treating salivary glands damaged by irradiation;or as a replacement or supplemental protein therapy. Secretion of anappropriate protein into the pancreatic juices is useful in preventingor controlling various lower gastrointestinal diseases, e.g. in treatingchronic infections of the stomach and/or intestinal tract; in treatingdegenerative pancreatic disorders; or as a replacement or supplementalprotein therapy (e.g., diabetes, intrinsic factor deficiency, digestiveenzyme deficiencies).

[0099] In a preferred embodiment, the proteins are secreted into theblood stream at levels sufficient for intravenous protein therapy. Forexample, the normal amount of a specific protein released into the bloodfrom the pancreas can be substantial, e.g. as much as 25% of the amountof duct-directed protein secretion. Blood stream levels of thetherapeutic protein may be enhanced by integration of multiple copies ofthe DNA of interest into the genome of the target cells, and/or byoperably linking a strong promoter (e.g., a promoter from CMV) and/orenhancer elements to the DNA of interest in the construct. Blood streamlevels may also be enhanced by implanting a greater number oftransformed cells (ex vivo gene therapy) or transformation of a greaternumber of target cells in the subject (in vivo gene therapy). Asdiscussed above, secretion of the therapeutic protein may also beenhanced by incorporating leader sequences, amino acid sequence motifs,or other elements which mediate intravenous-directed secretion into thesequence of the therapeutic protein.

[0100] Overall secretion from secretory glands is augmented by hormonalstimulation. For example, where the protein is primarily secreted intothe duct system and is secreted at lower levels into the blood stream,hormonal stimulation enhances both ductal and intravenous secretion.Thus, therapeutically effective levels of the protein in thegastrointestinal tract and the blood stream may be achieved or enhancedby administration of an appropriate, secretory gland specific hormone.For example, secretory gland secretion can be enhanced by administrationof a cholinergic agonist such as acetyl-β-methyl choline.

[0101] The actual number of transformed secretory gland cells requiredto achieve therapeutic levels of the protein of interest will varyaccording to several factors including the protein to be expressed, thelevel of expression of the protein by the transformed cells, the rate ofprotein secretion, the partitioning of the therapeutic protein betweenthe gastrointestinal tract and the blood stream, and the condition to betreated. For example, the desired intravenous level of therapeuticprotein can be readily calculated by determining the level of theprotein present in a normal subject (for treatment of a proteindeficiency), or by determining the level of protein required to effectthe desired therapeutic result. The level of expression of the proteinfrom transformed cells and the rate of protein secretion can be readilydetermined in vitro. Given the in vitro levels of protein expression andsecretion, and the estimated intravenous level of therapeutic proteindesired, the number of cells which should be transformed to effect thedesired levels can be readily calculated, and the gene therapy protocolcarried out accordingly.

[0102] A general schematic diagram showing the production of vectors andtransformed secretory gland cells according to the invention is providedin FIGS. 3A and 3B.

[0103] Assessment of Protein Therapy

[0104] Following either ex vivo or in vivo transfer of a DNA of interestinto a secretory gland, the effects of expression of the protein encodedby the DNA of interest can be monitored in a variety of ways. Generally,the presence of the protein in either a sample of blood, or a sample ofsaliva, pancreatic juices, urine, or mucosal secretions from the subjectcan be assayed for the presence of the therapeutic protein. Appropriateassays for detecting a protein of interest in either saliva or bloodsamples are well known in the art. For example, where secretory glandgene therapy has been performed to accomplish intravenous proteintherapy, a sample of blood can be tested for the presence of the proteinusing an antibody which specifically binds the therapeutic protein in anELISA assay. This assay can be performed either qualitatively orquantitatively. The ELISA assay, as well as other immunological assaysfor detecting a protein in a sample, are described in Antibodies: ALaboratory Manual (1988, Harlow and Lane, ed.s Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

[0105] Alternatively, or in addition, the efficacy of the proteintherapy can be assessed by testing a sample of blood, or saliva, orpancreatic juices or mucosal secretion for an activity associated withthe therapeutic protein (e.g., an enzymatic activity). For example,where the therapeutic protein has antimicrobial activity, the efficacyof therapy can be tested by examining the ability of the test sample toinhibit bacterial growth. Furthermore, the efficacy of secretory glandgene therapy can be assessed by monitoring the condition of themammalian subject for improvement. For example, where the therapeuticprotein is erythropoietin, the subject's blood is examined for ironcontent or other parameters associated with anemia. Where thetherapeutic protein is insulin, the efficacy of the therapy can beassessed by examining blood glucose levels of the mammalian subject orby measuring insulin (e.g., by using the human insulin radioimmunoassaykit, Linco Research Inc., St. Louis, Mo.).

EXAMPLES

[0106] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to carry out the invention and is not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperatures, etc.), but some experimental error and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1

[0107] In vivo Gene Transfer to Salivary Glands by Injection of NakedDNA Encoding Insulin

[0108] Four adult rats weighing approximately 300 g each wereanesthetized with an intraperitoneal injection of sodium pentobarbital.An incision was made parallel to the line of the mandible and bothparotid glands exposed by dissection. Two rats received a total of 100μl of 0.5 μg/μl pBAT14.hIns plasmid which contains CDNA encoding humaninsulin (FIG. 1), while the remaining two rats received 100 μl 0.9%saline (sham animals). The test and control samples were administered bymulti-site subcapsular injection to each parotid gland of each animal.No significant leakage of material or bleeding occurred. The wound wasclosed after administration. After 3 hours, the animals were awake,drinking water, and appearing normal.

[0109] Approximately 24 hours after cDNA injection, the animals wereagain anesthetized and a tracheostomy performed. A control blood samplewas drawn from the femoral vein of each animal. The cholinergic agonistacetyl-β-methyl choline (McH) was injected into each subcutaneously at0.8 mg/kg body weight into each animal. The salivary glands appearednormal and showed no signs of inflammation. Twenty minutes after McHinjection, saliva and blood samples were collected from each animal. Theblood samples were collected from the inferior vena cava and by heartpuncture. Serum was separated from the blood of all samples afterclotting, and kept at −20° C. prior to assay. In addition, blood wascollected from 10 normal rats, and serum prepared to determine the bloodlevel of insulin in untreated rats.

[0110] The results of this experiment are shown in Table 3. The level ofinsulin in the blood of transfected animals and in blood ofuntransfected animals was essentially the same. Administration of McHinduced an increase in serum insulin levels in both the control andtransfected animals. The concentration of insulin was higher after McHstimulation in the two transfected animals than in the McH-stimulatedcontrol animals. TABLE 3 Treatment Insulin (μU/ml) Normal (untreated)2.6 cDNA 1 −McH 1.4 +McH 10.0 cDNA 2 −McH 2.5 +McH 11.6 Control 1 −McH2.0 +McH 5.6 Control 2 −McH 2.0 +McH 9.2

Example 2

[0111] In vivo Gene Transfer to Salivary Glands by Injection of anIncreased Dosage Naked DNA Encoding Insulin

[0112] In a second experiment, four adult rats weighing approximately300 g each were anesthetized with an intraperitoneal injection of sodiumpentobarbital. Two rats received a total of 100 μl of 1-1.2 μg/μlpBAT14.hIns plasmid containing CDNA encoding human insulin, while theremaining two rats received 100 μl 0.9% saline (control animals). Thetest and control samples were administered by multi-site subcapsularinjection to each parotid gland of each animal as described above inExample 1, and the wound closed after administration.

[0113] Approximately 24 hours after injection, the animals were againanesthetized, blood samples drawn from each animal, and the cholinergicagonist McH administered at 0.8 mg/kg body weight by subcutaneousinjection. Twenty minutes after McH injection, blood samples were drawnfrom the inferior vena cava and by heart puncture of each animal.

[0114] Serum was separated from the blood of all samples after clotting,and kept at −20° C. prior to assay for insulin. The parotid glands ofall animals looked normal and showed no signs of inflammation.

[0115] As shown in Table 4, the levels of insulin in the blood of thetransfected animals was substantially higher than in the previousexperiment, suggesting that the increased dosage of CDNA resulted inincreased insulin production. Insulin levels in the transfected animalswere elevated by McH stimulation. Moreover, the animals transfected with100 μg-120 μg CDNA had about 50% greater insulin levels after McHstimulation than the animals transfected with 50 μg cDNA describedabove. TABLE 4 Treatment Insulin (μU/ml) cDNA 3 −McH 6.4 +McH 15.2 cDNA4 −McH 7.2 +McH 15.2

Example 3

[0116] Effects of Isoprenaline Stimulation on Expression of InsulinFollowing in vivo Transformation of Salivary Glands

[0117] Two transfected rats and two control rats were treated using thesame protocol, vector, and dose as in Example 2. Approximately 24 hoursafter injection, the animals were again anesthetized, blood samplesdrawn from each animal, and the adrenergic agonist isoprenaline (IsO)was administered at 0.1 μg/kg body weight by subcutaneous injection.Twenty minutes after Iso injection, blood samples were drawn from theinferior vena cava and by heart puncture of each animal. Serum wasseparated from the blood of all samples after clotting, and kept at −20°C. prior to assay for insulin.

[0118] As shown in Table 5, the blood insulin levels in the transfectedanimals was substantially elevated relative to control values. Injectionof Iso had no effect upon blood insulin levels. TABLE 5 TreatmentInsulin (μU/ml) cDNA 5 −IsO 8.5 +IsO 8.0 cDNA 6 −IsO 6.4 +IsO 8.0

Example 4

[0119] Effects of Streptozotocin on Insulin Levels in Rats Following invivo Transfer of CDNA Encoding Insulin to Salivary Glands

[0120] Streptozotocin, which induces diabetes mellitus in rats, wasadministered to three adults rats weighing approximately 220-230 g at 70mg/kg body weight by intraperitoneal injection. The animals wereanesthetized by intraperitoneal injection of sodium pentobarbital. Twoof the animals were injected with a 50 μl volume of 2 μg/μl pBAT14.hInsplasmid which contains cDNA encoding human insulin. The remaining ratreceived 100 μl 0.9% saline (control animal). The test and controlsamples were administered by multi-site subcapsular injection to eachparotid gland of each animal as described in Example 1.

[0121] Approximately 48 hours after cDNA or saline injection, theanimals were again anesthetized and a tracheostomy performed. A controlblood sample was drawn from the femoral vein of each animal. McH wasadministered at 0.8 mg/kg body weight by subcutaneous injection. Twentyminutes after McH injection, saliva and blood samples were collectedfrom each animal. The blood samples were collected from the inferior vencava and by heart puncture. Serum was separated from the blood of allsamples after clotting, and kept at −20° C. prior to assay. In addition,the salivary glands and a portion of the pancreas were removed andhomogenized in 50 mM phosphate buffer (pH 8.0) 1:10 w/v. The homogenateswere spun at 50,000×g for 1 h and the supernatant stored at −20° C. Asmall portion of parotid and salivary glands were fixed in 10% bufferedformalin and saved for histologic examination. The parotid glands showedno observable signs of inflammation as a results of CDNA injection.

[0122] As shown in Table 6, streptozotocin administration decreased theblood levels of insulin in the transfected animals. Stimulation with McHwas effective in increasing serum insulin levels in one of the twotransfected animals, but not in the control animal. TABLE 6 TreatmentInsulin (μU/ml) Glucose (mg/dl) cDNA −McH 0.5 268 +McH 8.8 385 cDNA −McH1.6 321 +McH 1.0 413 Control 1 −McH 5.2 230 +McH 4.8 335

Example 5

[0123] Summary of Results of in vivo Gene Transfer to Salivary Glands byPercutaneous Injection of Naked DNA Encoding Insulin

[0124] Nine adult rats were anesthetized with an intraperitonealinjection of sodium pentobarbital. Six rats were injected percutaneouslywith DNA encoding insulin. Two rats received a 100 μl volume of 0.5μg/μl pBAT14.hIns plasmid which contains CDNA encoding human insulin(low dose animals), while the four other transfected rats received a 100μl volume of 1.0 μg/μl pBAT14.hIns (high dose animals). The remainingthree rats received 100 μl 0.9% saline (sham animal).

[0125] Approximately 24 hours after injection, the animals were againanesthetized. Control blood samples were drawn from the femoral vein ofeach animal. Two of the control animals, two of the low dose animals,and two of the high dose animals received a subcutaneous injection of0.8 mg/kg body weight McH. Two of the high dose transfected animalsreceived a subcutaneous injection of the adrenergic agonist IsO at 0.1μg/kg body weight. Twenty minutes after McH or Iso injection, saliva andblood samples were collected from each animal. The blood samples werecollected from the inferior ven cava and by heart puncture. Serum wasseparated from the blood of all samples after clotting, and kept at −20°C. prior to assay.

[0126] The results of this experiment are shown in Table 7. The seruminsulin levels were highest in the high dose transfected animal group.The serum insulin levels of the low dose transfected group and thecontrol group were similar. After stimulation with McH, serum insulinlevels were again markedly higher in the high dose transfected groupthan in either the low dose transfected or the control groups. Seruminsulin levels after McH stimulation were higher in the low dosetransfected group than in the control group. Iso injection of high dosetransfected rats had no significant effect upon serum insulin levels ascompared to serum insulin levels in the absence of agonist. These datashow that high dose CDNA increase both unstimulated (—McH) andMcH-stimulated insulin responses. TABLE 7 Average values for the effectpercutaneous administration of human insulin cDNA to parotid gland onserum insulin Treatment Insulin (μU/ml) Control  2.5 (12) Low dose cDNA(0.5 μg/μl)  2.0 (2) High dose cDNA (1.0 μg/μl)  7.4 (6) Withcholinergic stimulation (McH) Control  7.6 (2) Low dose cDNA (0.5 μg/μl)13.0 (2) High dose cDNA (1.0 μg/μl) 15.2 (2)

Example 6

[0127] Ex vivo Gene Transfer of DNA Encoding Erythropoietin into a HumanSalivary Gland

[0128] A biopsy of a human parotid gland is obtained by surgicalincision and extraction of a small (1 cm²) tissue sample. The tissuesample is passed through a sterile 1 mm² mesh screen to provide a singlecell suspension of salivary gland cells. The cells are transferred to atissue culture flask and incubated in complete tissue culture medium ina tissue culture incubator at 37° C., 5% CO₂. When the flask reachesapproximately 70% to 90% confluency, the cells are removed from theflask by incubation in sterile phosphate-buffered saline (PBS)containing EDTA. The cells are then split into two flasks and incubationis continued. After these cells reach confluency in the flasks, theculture medium is replaced with medium containing liposomes containingDNA encoding erythropoietin. The erythropoietin DNA is operably linkedto the salivary amylase promoter and is additionally linked to a geneencoding neomycin resistance. The cells are incubated for 24 hrs in thepresence of the liposome-containing medium. The medium is then removedand replaced with fresh medium containing neomycin. After an additionaltwo to three days incubation, the cells are again split into two flasksand grown to confluency.

[0129] To determine whether the transformed cells are producing andsecreting erythropoietin, a sample of the cell culture medium isexamined by Western blot. Briefly, the cell supernatant sample iscollected and concentrated by ultrafiltration using a filter whichallows filtration of proteins of only an appropriate molecular weightrange. Protein concentration is determined using Lowry's method (Lowryet al., J. Biol. Chem. 193:265-275, 1951). The samples are thensubjected to polyacrylamide gel electrophoresis. The size of theprotein(s) is estimated using prestained protein molecular weightstandards. the electrophoresed proteins are transferred tonitrocellulose membranes and the transferred proteins detected byWestern blotting (Towbin et al., Proc. Natl. Acad. Scl. USA76:4350-4354, 1979) using a mixture of mouse antisera against theprotein encoded by the DNA of interest. The immunoblots are developed byincubating the antibodies with goat anti-mouse IgG conjugated toalkaline phosphatase. The conjugate is if made visible by reaction ofthe immunoblot with the chromogenic substrate5-bromo-4-chloro-3-indolyl-phosphate/p-nitro blue tetrazolium chloride.Expression and secretion of erythropoietin by the transformed cells iscorrelated with the presence of a protein band of appropriate molecularweight on the immunoblot.

[0130] The transformed cells are then removed from the flasks andimplanted into the parotid gland of the patient. The establishment andacceptance of the implant is monitored at least daily. The expression oferythropoietin by the implant and the secretion of erythropoietin intothe blood stream of the patient are determined by ELISA. Briefly, asample of blood is collected and the sample subjected to centrifugation.The serum is then collected and applied to a microtiter plate havingbound to its surface an anti-erythropoietin antibody. The wells of theplate are then washed with buffer, and a second anti-erythropoietinantibody is applied to the wells. Following incubation, the wells areagain washed to rid of excess second antibody, and an antibody whichspecifically binds the second, fluorescently labeled anti-erythropoietinantibody is applied to the wells. After a final incubation and washingstep to rid of unbound material, the label is then detected with anELISA reader. Detection of a fluorescent signal is indicative of thepresence of erythropoietin in the blood sample and thus the expressionand secretion of erythropoietin by the implanted cells.

Example 7

[0131] In vivo Gene Transfer of DNA Encoding Clotting Factor VIII byOral Infection with a Replication-Deficient Adenovirus

[0132] A DNA fragment containing the DNA sequence encoding clottingfactor VIII (Gitschier et al. Nature, 312:326-330, 1984; Wood et al.Nature, 312:330-337, 1984) is operably linked to the moloney-LTRpromoter. This promoter-clotting factor VIII cassette is inserted into areplication-deficient recombinant adenovirus vector. The adenovirusvector has been constructed so that the 5′ end of the E1 promoter and aportion of the E3 regions are deleted. The promoter-clotting factor VIIIDNA cassette is inserted at the site of the El deletion. Adenovirusvectors are prepared, purified, and titered as previously described(Rosenfeld et al. Science 252:431-434, 1991; Rosenfeld et al., Cell68:143-155, 1992).

[0133] Approximately 5×10⁹ pfu (plaque-forming units) of the recombinantvirus containing clotting factor VIII DNA are orally administered.Expression of the clotting factor VIII DNA and intravenous secretion ofclotting factor VIII are assessed-as described above.

Example 8

[0134] In vivo Gene Transfer of DNA Encoding Human Growth Hormone byRetrograde Injection of DNA

[0135] A DNA fragment encoding human growth hormone (hGH) is operablylinked to the LTR of Rous sarcoma virus, which serves as a promoter, andthe SV40 type T antigen, which serves as a nuclear localization signal.This promoter-localization signal-hGH DNA cassette is then inserted intothe bacterial plasmid pBR322. Escherichia coli is then transformed withthe plasmid using conventional transformation procedures. E. colicontaining the plasmid are selected by virtue of the tetracycline orampicillin resistance encoded by pBR322, and the transformed bacterialcells propagated in culture. Plasmid DNA is then isolated from thetransformed bacterial cell culture and the DNA purified by cesiumgradient.

[0136] Approximately 10 mg to 20 mg of the purified plasmid DNAcontaining hGH DNA is injected into the salivary gland of a humanpatient by retrograde injection via a salivary gland duct. Expressionand intravenous secretion of the protein is assessed using the methoddescribed above.

Example 9

[0137] In vivo Gene Transfer of DNA Encoding Human Growth Hormone byRetrograde Injection of DNA into the Pancreas

[0138] Four constructs for expression of human growth hormone (hGH) wereprepared using techniques well known in the art (see, for example,Sambrook et al. ibid). The first construct, pFGH, contains the genomichGH DNA sequence inserted in the commercially available vectorpBLUESCRIPT SK+^(TM) (Stratagene, LaJolla, Calif.) (FIG. 4). Because thehGH coding sequence is not linked to a promoter, this vector providesfor no or only low-level hGH expression. Thus, the pFGH construct servesas a negative control for hGH expression in the pancreas. The secondconstruct, PFGH.CMV, was constructed by operably inserting the promoterfrom the immediate early gene of human CMV upstream of the genomic hGHsequence of the pFGH vector (FIG. 5). The third construct, pFGH.chymo,was constructed by operably inserting the rat chymotrypsin B genepromoter upstream of the genomic hGH sequence of the pFGH vector (FIG.6). The fourth construct, pFGH.RSV, was constructed by operablyinserting the promoter from the long terminal repeat (LTR) of RSVupstream of the genomic hGH sequence of the pFGH vector.

[0139] Each of the four vectors was used to transfect the pancreas ofapproximately 300 g adult rats (pFGH+lipofectin, 4 rats;pFGH.chymo+lipofectin, 4 rats; pFGH.RSV+lipofectin, 4 rats;pFGH.CMV+lipofectin, 10 rats; PFGH.CMV without lipofectin, 7 rats;negative control (no DNA, no lipofectin), 3 rats). Pancreatictransfection was accomplished by first anesthetizing the rats andperforming a laparotomy to expose the duodenum. The pancreas and theassociated common bile duct were identified, and the common bile ductwas cannulated either extraduodenally or through the papilla of Vater.The hepatic duct was occluded, and 100 μl of phosphate-buffered saline(PBS) containing one of the four vectors, or 100 μl of PBS alone as anegative control, were slowly injected or infused into the pancreaticduct in a retrograde direction. The vector-containing solutions werecomposed of 8 μg DNA per 100 μl in PBS, either with or without 6%lipofectin, a cationic lipid used to increase transfection efficiency.The solution was left in place for 5 min before secretory flow wasallowed to resume and hepatic duct blockage removed. The catheter wasleft in place and inserted into the duodenum through a small hole toensure adequate biliary and pancreatic flow post-operatively. Theabdomen was then closed with sutures. The animals recovered fully andrapidly from the surgery without obvious side effects. This transfectionmethod provides direct access of the vector to over 90% of thepancreatic gland cells.

[0140] At 48 hr after surgery, a blood sample was obtained to measureserum hGH levels, and the rats were sacrificed. At autopsy, the pancreasof both control and test rats appeared normal, and exhibited no gross ormicroscopic pathology.

[0141] The pancreas was dissected free from the mesenteric suface andwas homogenized in cold 0.2 M (pH 8.0) sodium phosphate buffer (1:10w/v) containing protease inhibitors aprotinin, leupeptin, pepstatin, andPEFABLOC SC^(TM). Homogenization was completed by shearing after 10passes with a motorized pestle at approximately 4000 rpm in a glasshomogenizer. The homogenate was then centrifuged at 1000 g for 15 min.The supernatant was collected and stored at −80° C. until analysis. Thelevels of hGH in the serum and pancreatic protein samples were measuredusing the hGH radioimmune assay (Nichols Institute). Each assay wasperformed in duplicate and compared to a set of control samples.

[0142] Rats injected with the pFGH.CMV vector expressed higher levels ofhGH in the pancreatic tissue (FIG. 7), compared to background levels ofpancreatic hGH expression in rats injected with either no DNA (PBSalone) or the pFGH vector (hGH DNA with no promoter). The addition oflipofectin modestly increased hGH expression in rats injected with thepFGH.CMV construct. In addition, rats transfected with the pFGH.CMVvector secreted hGH in the serum at levels increased relative to hGHsecretion levels in rats injected with either control samples (no DNA orpFGH, or with samples containing hGH DNA linked to either thechymotrypsin B or RSV promoters (FIG. 8). In FIG. 9, all data from theabove experiments (including all controls and vectors) are analyzed byplotting the hGH serum levels against the hGH tissue levels. This graphshows that higher tissue levels result in higher levels of secretioninto the blood. Thus, retrograde pancreatic injection of the PFGH.CMVvector successfully transfected pancreatic cells to provide both hGHpancreatic tissue expression and hGH secretion into the bloodstream.

Example 10

[0143] In vivo gene Transfer of DNA Encoding Intrinsic Factor byCannulation of Naked DNA into the Pancreas

[0144] DNA encoding intrinsic factor (Hewitt et al. Genomics 10:432-440,1991) is operably linked to the pancreatic α-amylase promoter. Thispromoter-intrinsic factor DNA cassette is then inserted into a plasmidcapable of replicating in Escherichia coli. The plasmid construct isthen used to transform E. coli, the transformed cells are expanded, andthe construct DNA purified. The purified DNA is then resuspended in 0.9%saline and a volume of the DNA solution is administered to a humanpatient suffering from pernicious anemia. Approximately 100 mg to 200 mgof DNA is delivered to the pancreas of the patient by cannulation of thepancreatic duct by duodenal intubation using endoscopic retrogradecholangio-pancreatography. Expression and secretion of intrinsic factorinto the gastrointestinal tract is assessed using the protocol describedabove. The efficacy of the therapy is also assessed by examining thelevel of vitamin B₁₂ in the patient's blood.

Example 11

[0145] In vivo Gene Transfer of DNA Encoding Human Growth Hormone byCannulation of Naked DNA into the Pancreas

[0146] DNA encoding human growth hormone (Marshall et al., Biotechnology24:293-298, 1992) is operably linked to the human insulin promoter. Thispromoter-human growth hormone DNA cassette is then inserted into aplasmid capable of replicating in Escherichia coli. The plasmidconstruct is then used to transform E. coli, the transformed cells areexpanded, and the construct DNA purified. The purified DNA is thenresuspended in 0.9% saline and a volume of the DNA solution isadministered to a human patient. Approximately 100 mg to 200 mg of DNAis delivered to the pancreas of the patient by cannulation of thepancreatic duct by duodenal intubation using endoscopic retrogradecholangio-pancreatography. Expression and secretion of human growthhormone into the blood stream is assessed by detection of the protein inthe patient's blood.

Example 12

[0147] In vivo Gene Transfer of DNA Encoding Human Insulin byCannulation of Naked DNA into the Pancreas

[0148] DNA encoding human insulin is operably linked to the pancreaticα-amylase promoter. This promoter-human insulin DNA cassette is theninserted into a plasmid capable of replicating an Escherichia coli. Theplasmid construct is then used to transform E. coli, the transformedcells are expanded, and the construct DNA purified. The purified DNA isthen resuspended in 0.9% saline and a volume of the DNA solution isadministered to a human patient suffering from an insulin deficiency(e.g., diabetes). Approximately 100 mg to 200 mg of DNA is delivered tothe pancreas of the patient by cannulation of the pancreatic duct byduodenal intubation using endoscopic retrogradecholangio-pancreatography. Expression and secretion of insulin into theblood stream is assessed by examining blood glucose levels or bymeasuring insulin (e.g., by using a human insulin radioimmunoassay kit.

[0149] Following procedures similar to those described above, othertherapeutic proteins can be expressed from DNA inserted in the genome ofa salivary gland cell by gene transfer according to the invention.

[0150] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A method of treatment, comprising: geneticallytransforming cells of a secretory gland of a mammalian patient with aconstruct comprising a DNA of interest which expresses a protein thatthe patient is in need of and a eukaryotic promoting sequence operablylinked to the DNA of interest; and allowing the genetically transformedcells to express the protein in a therapeutically effective amountthereby treating the patient.
 2. The method of claim 1 , wherein thesecretory gland is selected from the group consisting of the salivarygland and the pancreas.
 3. The method of claim 2 , wherein the salivarygland is a parotid gland.
 4. The method of claim 1 , wherein the proteinis secreted into the blood stream and the protein is selected from thegroup consisting of: insulin, growth hormone, clotting factor VIII, anderythropoietin.
 5. The method of claim 1 , wherein the mammalian patientis a human the protein is a human protein and the secretory gland is apancreas.
 6. The method of claim 1 , wherein the protein is human growthhormone and the secretory gland is a pancreas.
 7. The method of claim 6, where the cells of the pancreas are transformed in vivo by injecting asolution comprising vectors which vectors comprise DNA expressing humangrowth hormone.
 8. The method of claim 6 , wherein the mammalian patientis a human and the protein is human clotting factor VIII.
 9. The methodof claim 6 , wherein the mammalian patient is a human and the protein ishuman intrinsic factor.
 10. The method of claim 6 , wherein themammalian patient is a human and the protein is human erythropoietin.11. The method of claim 1 , wherein the promoter sequence is an amylasepromoter.
 12. The method of claim 11 , wherein the secretory gland isthe pancreas and the amylase promoter is a pancreatic α-amylasepromoter.
 13. The method of claim 11 , wherein the secretory gland is asalivary gland and the amylase promoter is a salivary α-amylasepromoter.
 14. The method of claim 1 , wherein the DNA of interest andpromoter are incorporated into a viral vector.
 15. The method of claim 5, wherein the protein is secreted into the gastrointestinal tract. 16.The method of claim 6 , wherein the protein is secreted into'thepatient's saliva.
 17. A genetically transformed secretory gland cell,comprising: a DNA of interest which expresses a therapeuticallyeffective protein which DNA is artificially and operatively inserted inthe genome of the cell; a promoter operatively linked to the DNA. 18.The cell of claim 17 , wherein the promoter is a amylase promoter. 19.The cell of claim 17 , wherein the cell is a human salivary gland celland the promoter is a human salivary amylase promoter.
 20. The cell ofclaim 17 , wherein the cell is a human pancreatic cell and the promoteris a human pancreatic α-amylase promoter.